Vectors with modified initiation codon for the translation of aav-rep78 useful for production of aav

ABSTRACT

The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AA V) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. patentapplication Ser. No. 15/424,560 filed Feb. 3, 2017, which is aContinuation Application of U.S. patent application Ser. No. 14/603,469,filed Jan. 23, 2015, now U.S. Pat. No. 9,708,627, which is aContinuation Application of U.S. patent application Ser. No. 13/945,505,filed Jul. 18, 2013, now U.S. Pat. No. 8,952,144, which is a DivisionalApplication of U.S. patent application Ser. No. 12/306,239, filed Dec.22, 2008, now U.S. Pat. No. 8,512,981, which is the National Phase ofInternational Patent Application No. PCT/NL2007/050298, filed Jun. 20,2007, published on Dec. 27, 2007 as WO/2007/148971 A1, which claimspriority to European Patent Application No. 06115804.4, filed Jun. 21,2006, which claims priority to U.S. Provisional Application No.60/815,262, filed Jun. 21, 2006. The contents of these applications areherein incorporated by reference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 3, 2018, isnamed 069818-2522Sequence.txt and is 17 KB.

FIELD OF THE INVENTION

The present invention relates to the production of adeno-associated vimsin insect cells and to adeno-associated vims with improvements inexpression and stability of the viral rep proteins that increase theproductivity of adeno-associated viral vectors in insect cells.

BACKGROUND OF THE INVENTION

Adeno-associated vims (AAV) may be considered as one of the mostpromising viral vectors for human gene therapy. AAV has the ability toefficiently infect dividing as well as non-dividing human cells, the AAVviral genome integrates into a single chromosomal site in the hostcell's genome, and most importantly, even though AAV is present in manyhumans it has never been associated with any disease. In view of theseadvantages, recombinant adeno-associated vims (rAAV) is being evaluatedin gene therapy clinical trials for hemophilia B, malignant melanoma,cystic fibrosis, and other diseases.

Host cells that sustain AAV replication in vitro are all derived frommammalian cell types. Therefore, rAAV for use in gene therapy has thusfar mainly been produced on mammalian cell lines such as e.g. 293 cells,COS cells, HeLa cells, KB cells, and other mammalian cell lines (seee.g. U.S. Pat. No. 6,156,303, U.S. Pat. No. 5,387,484, U.S. Pat. No.5,741,683, U.S. Pat. No. 5,691,176, U.S. Pat. No. 5,688,676, US20020081721, WO 00/47757, WO 00/24916, and WO 96/17947). rAAV vectorsare typically produced in such mammalian cell culture systems byproviding DNA plasmids that contain the therapeutic gene flanked by theorigin of AAV replication (inverted terminal repeats or ITRs), genes forAAV replication proteins Rep78, Rep68, Rep52, and Rep40, and genes forvirion or structural proteins VPl, VP2, and VP3. In addition, a plasmidcontaining early genes from adenovims (E2A, E40RF6, VARNA) is providedto enhance the expression of the AAV genes and improve vector yield (seee.g. Grimm et al., 1998, Hum. Gene Ther. 9:2745-2760). However, in mostof these mammalian cell culture systems, the number of AAV particlesgenerated per cell is on the order of 10⁴ particles (reviewed in Clark,2002, Kidney Int. 61(Suppl. 1):9-15). For a clinical study, more than10¹⁵ particles of rAAV may be required. To produce this number of rAAVparticles, transfection and culture with approximately 10¹¹ culturedhuman 293 cells, the equivalent of 5,000 175-cm² flasks of cells, wouldbe required, which means transfecting up to 10¹¹ 293 cells. Therefore,large scale production of rAAV using mammalian cell culture systems toobtain material for clinical trials has already proven to beproblematic, production at commercial scale may not even be feasible.Furthermore there is always the risk, that a vector for clinical usethat is produced in a mammalian cell culture will be contaminated withundesirable, perhaps pathogenic, material present in the mammalian hostcell.

To overcome these problems of mammalian productions systems, recently,an AAV production system has been developed using insect cells (Urabe etal., 2002, Hum. Gene Ther. 13:1935-I943; US 20030I48506 and US 20040I97895). For production of AAV in insect cells some modifications werenecessary in order to achieve the correct stoichiometry of the three AAVcapsid proteins (VPI, VP2 and VP3), which relies on a combination ofalternate usage of two splice acceptor sites and the suboptimalutilization of an ACG initiation codon for VP2 that is not accuratelyreproduced by insect cells. To mimic the correct stoichiometry of thecapsid proteins in insect cells Urabe et al. (2002, supra) use aconstruct that is transcribed into a single polycistronic messenger thatis able to express all three VP proteins without requiring splicing andwherein the most upstream initiator codon is replaced by the suboptimalinitiator codon ACG. In co-pending application (PCT/NL2005/0500I8) thepresent inventors have further improved the infectivity ofbaculovirus-produced rAAV vectors based production by furtheroptimisation of the stoichiometry of AAV capsid proteins in insectcells.

For expression of the AAV Rep proteins in the AAV insect cell expressionsystem as initially developed by Urabe et al. (2002, supra), arecombinant baculovirus construct is used that harbours two independentRep expression units (one for Rep78 and one for Rep52), each under thecontrol of a separate insect cell promoter, the MEI and PolH promoters,respectively. In this system, the MEI promoter, a much weaker promoterthan the PolH promoter, was chosen for driving Rep78 expression since itis known that in mammalian cells a less abundant expression of Rep78 ascompared to Rep52 favours high vector yields (Li et al., I997, J Virol.7I:5236-43; Grimm et al., I998, supra).

More recently however, Kohlbrenner et al. (2005, Mol. Ther. I2:I2I7-25)reported that the baculovirus construct for expression of the two Repprotein, as used by Urabe et al., suffers from an inherent instability.By splitting the palindromic orientation of the two Rep genes in Urabe'soriginal vector and designing two separate baculovirus vectors forexpressing Rep52 and Rep78, Kohlbrenner et al. (2005, supra) increasedthe passaging stability of the vector. However, despite the consistentexpression of Rep78 and Rep52 from the two independent baculovirus-Repconstructs in insect cells over at least 5 passages, rAAV vector yieldis 5 to 10-fold lower as compared to the original baculovirus-Repconstruct designed by Urabe et al. (2002, supra).

There is thus still a need to overcome the above serious limitations oflarge scale (commercial) production of AAV vectors in insect cells. Thusit is an object of the present invention to provide for means andmethods that allow for stable and high yield (large scale) production ofAAV vectors in insect cells.

DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide (or polypeptide) elements in a functional relationship. Anucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, atranscription regulatory sequence is operably linked to a codingsequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein encoding regions,contiguous and in reading frame.

“Expression control sequence” refers to a nucleic acid sequence thatregulates the expression of a nucleotide sequence to which it isoperably linked. An expression control sequence is “operably linked” toa nucleotide sequence when the expression control sequence controls andregulates the transcription and/or the translation of the nucleotidesequence. Thus, an expression control sequence can include promoters,enhancers, internal ribosome entry sites (IRES), transcriptionterminators, a start codon in front of a protein-encoding gene, splicingsignal for introns, and stop codons. The term “expression controlsequence” is intended to include, at a minimum, a sequence whosepresence are designed to influence expression, and can also includeadditional advantageous components. For example, leader sequences andfusion partner sequences are expression control sequences. The term canalso include the design of the nucleic acid sequence such thatundesirable, potential initiation codons in and out of frame, areremoved from the sequence. It can also include the design of the nucleicacid sequence such that undesirable potential splice sites are removed.It includes sequences or polyadenylation sequences (pA) which direct theaddition of a polyA tail, i.e., a string of adenine residues at the3′-end of a mRNA, sequences referred to as polyA sequences. It also canbe designed to enhance mRNA stability. Expression control sequenceswhich affect the transcription and translation stability, e.g.,promoters, as well as sequences which effect the translation, e.g.,Kozak sequences, are known in insect cells. Expression control sequencescan be of such nature as to modulate the nucleotide sequence to which itis operably linked such that lower expression levels or higherexpression levels are achieved.

As used herein, the term “promoter” or “transcription regulatorysequence” refers to a nucleic acid fragment that functions to controlthe transcription of one or more coding sequences, and is locatedupstream with respect to the direction of transcription of thetranscription initiation site of the coding sequence, and isstructurally identified by the presence of a binding site forDNA-dependent RNA polymerase, transcription initiation sites and anyother DNA sequences, including, but not limited to transcription factorbinding sites, repressor and activator protein binding sites, and anyother sequences of nucleotides known to one of skill in the art to actdirectly or indirectly to regulate the amount of transcription from thepromoter. A “constitutive” promoter is a promoter that is active in mosttissues under most physiological and developmental conditions. An“inducible” promoter is a promoter that is physiologically ordevelopmentally regulated, e.g. by the application of a chemicalinducer. A “tissue specific” promoter is only active in specific typesof tissues or cells.

The terms “substantially identical”, “substantial identity” or“essentially similar” or “essential similarity” means that two peptideor two nucleotide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default parameters, share at least acertain percentage of sequence identity as defined elsewhere herein. GAPuses the Needleman and Wunsch global alignment algorithm to align twosequences over their entire length, maximizing the number of matches andminimizes the number of gaps. Generally, the GAP default parameters areused, with a gap creation penalty=50 (nucleotides)/8 (proteins) and gapextension penalty=3 (nucleotides)/2 (proteins). For nucleotides thedefault scoring matrix used is nwsgapdna and for proteins the defaultscoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89,915-919). It is clear than when RNA sequences are said to be essentiallysimilar or have a certain degree of sequence identity with DNAsequences, thymine (T) in the DNA sequence is considered equal to uracil(U) in the RNA sequence. Sequence alignments and scores for percentagesequence identity may be determined using computer programs, such as theGCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685Scranton Road, San Diego, Calif. 92121 USA or the open-source softwareEmboss for Windows (current version 2.7.1-07). Alternatively percentsimilarity or identity may be determined by searching against databasessuch as PASTA, BLAST, etc.

Nucleotide sequences encoding parvoviral Rep proteins of the inventionmay also be defined by their capability to hybridise with the nucleotidesequence of SEQ ID NO.10, respectively, under moderate, or preferablyunder stringent hybridisation conditions. Stringent hybridisationconditions are herein defined as conditions that allow a nucleic acidsequence of at least about 25, preferably about 50 nucleotides, 75 or100 and most preferably of about 200 or more nucleotides, to hybridiseat a temperature of about 65° C. in a solution comprising about 1 Msalt, preferably 6×SSC or any other solution having a comparable ionicstrength, and washing at 65° C. in a solution comprising about 0.1 Msalt, or less, preferably 0.2×SSC or any other solution having acomparable ionic strength. Preferably, the hybridisation is performedovernight, i.e. at least for 10 hours and preferably washing isperformed for at least one hour with at least two changes of the washingsolution. These conditions will usually allow the specific hybridisationof sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridizationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates the use of animal parvoviruses, inparticular dependoviruses such as infectious human or simian AAV, andthe components thereof (e.g., an animal parvovirus genome) for use asvectors for introduction and/or expression of nucleic acids in mammaliancells. In particular the invention relates to improvements inproductivity of such parvoviral vectors when produced in insect cells.

Viruses of the Parvoviridae family are small DNA animal viruses. Thefamily Parvoviridae may be divided between two subfamilies: theParvovirinae, which infect vertebrates, and the Densovirinae, whichinfect insects. Members of the subfamily Parvovirinae are hereinreferred to as the parvoviruses and include the genus Dependovirus. Asmay be deduced from the name of their genus, members of the Dependovirusare unique in that they usually require coinfection with a helper virussuch as adenovirus or herpes virus for productive infection in cellculture. The genus Dependovirus includes AAV, which normally infectshumans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g.,serotypes 1 and 4), and related viruses that infect other warm-bloodedanimals (e.g., bovine, canine, equine, and ovine adeno-associatedviruses). Further information on parvoviruses and other members of theParvoviridae is described in Kenneth I. Berns, “Parvoviridae: TheViruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed.1996). For convenience the present invention is further exemplified anddescribed herein by reference to AAV. It is however understood that theinvention is not limited to AAV but may equally be applied to otherparvoviruses.

The genomic organization of all known AAV serotypes is very similar. Thegenome of AAV is a linear, single-stranded DNA molecule that is lessthan about 5,000 nucleotides (nt) in length. Inverted terminal repeats(ITRs) flank the unique coding nucleotide sequences for thenon-structural replication (Rep) proteins and the structural (VP)proteins. The VP proteins (VPl, -2 and -3) form the capsid. The terminal145 nt are self-complementary and are organized so that an energeticallystable intramolecular duplex forming a T-shaped hairpin may be formed.These hairpin structures function as an origin for viral DNAreplication, serving as primers for the cellular DNA polymerase complex.Following wtAAV infection in mammalian cells the Rep genes (i.e. Rep78and Rep52) are expressed from the P5 promoter and the P19 promotor,respectively and both Rep proteins have a function in the replication ofthe viral genome. A splicing event in the Rep ORF results in theexpression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 andRep40). However, it has been shown that the unspliced mRNA, encodingRep78 and Rep52 proteins, in mammalian cells are sufficient for AAVvector production. Also in insect cells the Rep78 and Rep52 proteinssuffice for AAV vector production.

A “recombinant parvoviral or AAV vector” (or “rAAV vector”) hereinrefers to a vector comprising one or more polynucleotide sequences ofinterest, genes of interest or “transgenes” that are flanked byparvoviral or AAV inverted terminal repeat sequences (ITRs). Such rAAVvectors can be replicated and packaged into infectious viral particleswhen present in an insect host cell that is expressing AAV rep and capgene products (i.e. AAV Rep and Cap proteins). When an rAAV vector isincorporated into a larger nucleic acid construct (e.g. in a chromosomeor in another vector such as a plasmid or baculovirus used for cloningor transfection), then the rAAV vector is typically referred to as a“pro-vector” which can be “rescued” by replication and encapsidation inthe presence of AAV packaging functions and necessary helper functions.

In a first aspect the invention relates to a nucleotide sequencecomprising an open reading frame comprising nucleotide sequencesencoding animal parvoviruses Rep proteins, wherein the initiation codonfor translation of the parvoviral Rep78 protein is a suboptimalinitiation codon. The suboptimal initiation codon preferably is aninitiation codon that effects partial exon skipping. Partial exonskipping is herein understood to mean that at least part of theribosomes do not initiate translation at the suboptimal initiation codonof the Rep78 protein but at an

initiation codon further downstream, whereby preferably the initiationcodon further downstream is the initiation codon of the Rep52 protein.The suboptimal initiation codon preferably effects partial exon skippingupon expression of the nucleotide sequence in an insect cell.Preferably, the suboptimal initiation codon effects partial exonskipping in an insect cell so as to produce in the insect cell a molarratio of Rep78 to Rep52 in the range of 1:10 to 10:1, 1:5 to 5:1, or 1:3to 3:1, preferably at about 20-40 hours post infection, more preferablyat about 30-40 hours post infection, using a baculovirus expression. Themolar ration of the Rep78 and Rep52 may be determined by means ofWestern blotting as described in Example 1.1.3, preferably using amonoclonal antibody that recognizes a common epitope of both Rep78 andRep52, or using the antibody described in Example 1.1.3.

The term “suboptimal initiation codon” herein not only refers to thetri-nucleotide initiation codon itself but also to its context. Thus, asuboptimal initiation codon may consist of an “optimal” ATG codon in asuboptimal context, e.g. a non-Kozak context. However, more preferredare suboptimal initiation codons wherein the tri-nucleotide initiationcodon itself is suboptimal, i.e. is not ATG. Suboptimal is hereinunderstood to mean that the codon is less efficient in the initiation oftranslation in an otherwise identical context as compared to the normalATG codon. Preferably, the efficiency of suboptimal codon is less than90, 80, 60, 40 or 20% of the efficiency of the normal ATG codon in anotherwise identical context. Methods for comparing the relativeefficiency of initiation of translation are known per se to the skilledperson. Preferred suboptimal initiation codons may be selected from ACG,TTG, CTG, and GTG. More preferred is ACG.

A nucleotide sequence encoding animal parvoviruses Rep proteins, isherein understood as a nucleotide sequence encoding the non-structuralRep proteins that are required and sufficient for parvoviral vectorproduction in insect cells such the Rep78 and Rep52 proteins. The animalparvovirus nucleotide sequence preferably is from a dependovirus, morepreferably from a human or simian adeno-associated virus (AAV) and mostpreferably from an AAV which normally infects humans (e.g., serotypes 1,2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4). Anexample of a nucleotide sequence encoding animal parvoviruses Repproteins is given in SEQ ID No.IO, which depicts a part of the AAVserotype-2 sequence genome encoding the Rep proteins. The Rep78 codingsequence comprises nucleotides 11-1876 and the Rep52 coding sequencecomprises nucleotides 683-1876. It is understood that the exactmolecular weights of the Rep78 and Rep52 proteins, as well as the exactpositions of the translation initiation codons may differ betweendifferent parvoviruses. However, the skilled person will know how toidentify the corresponding position in nucleotide sequence from otherparvoviruses than AAV-2. A nucleotide sequence encoding animalparvoviruses Rep proteins may thus also be defined as a nucleotidesequence:

-   -   a) that encodes a polypeptide comprising an amino acid sequence        that has at least 50, 60, 70, 80, 88, 89, 90, 95, 97, 98, or 99%        sequence identity with the amino acid sequence of SEQ ID NO. 11;    -   b) that has at least 50, 60, 70, 80, 81, 82, 85, 90, 95, 97, 98,        or 99% sequence identity    -   with the nucleotide sequence of positions 11-1876 of SEQ ID NO.        10;    -   c) the complementary strand of which hybridises to a nucleic        acid molecule sequence of or (b);    -   d) nucleotide sequences the sequence of which differs from the        sequence of a nucleic acid molecule of (c) due to the degeneracy        of the genetic code.

Preferably, the nucleotide sequence encodes animal parvovimses Repproteins that are required and sufficient for parvoviral vectorproduction in insect cells.

A further preferred nucleotide sequence of the invention comprises anexpression control sequence that comprising a nine nucleotide sequenceof SEQ. ID NO:7 or a nucleotide sequence substantially homologous toSEQ. ID NO:7, upstream of the initiation codon of the nucleotidesequence encoding the parvoviral Rep78 protein. A sequence withsubstantial identity to the nucleotide sequence of SEQ. ID NO:7 and thatwill help increase expression of the parvoviral Rep78 protein is e.g. asequence which has at least 60%, 70%, 80% or 90% identity to the ninenucleotide sequence of SEQ ID NO:7.

Elimination of possible false translation initiation sites in the Repprotein coding sequences, other than the Rep78 and Rep52 translationinitiation sites, of other parvovimses will be well understood by anartisan of skill in the art, as will be the elimination of putativesplice sites that may be recognized in insect cells. The variousmodifications of the wild-type parvoviral sequences for properexpression in insect cells is achieved by application of well-knowngenetic engineering techniques such as described e.g. in Sambrook andRussell (2001) “Molecular Cloning: A Laboratory Manual (3rd edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NewYork. Various further modifications of Rep protein coding regions areknown to the skilled artisan which could increase yield of Rep protein.These modifications are within the scope of the present invention.

In a further aspect the invention relates to a nucleic acid constructcomprising a nucleotide sequence encoding parvoviral Rep proteins asdefined above. Preferably, in the construct, the nucleotide sequenceencoding the parvoviral Rep proteins is operably linked to expressioncontrol sequences for expression in an insect cell. These expressioncontrol sequences will at least include a promoter that is active ininsect cells. Techniques known to one skilled in the art for expressingforeign genes in insect host cells can be used to practice theinvention. Methodology for molecular engineering and expression ofpolypeptides in insect cells is described, for example, in Summers andSmith. 1986. A Manual of Methods for Baculovirus Vectors and InsectCulture Procedures, Texas Agricultural Experimental Station Bull. No.7555, College Station, Tex.; Luckow. 1991. In Prokop et al., Cloning andExpression of Heterologous Genes in Insect Cells with BaculovirusVectors' Recombinant DNA Technology and Applications, 97-152; King, L.A. and R. D. Possee, 1992, The baculovirus expression system, Chapmanand Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow,1992, Baculovirus Expression Vectors: A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D., 1995, Baculovirus ExpressionProtocols, Methods in Molecular Biology, volume 39; U.S. Pat. No.4,745,051; US2003148506; and WO 03/074714. A particularly suitablepromoter for transcription of the nucleotide sequence of the inventionencoding of the parvoviral Rep proteins is e.g. the polyhedron promoter.However, other promoters that are active in insect cells are known inthe art, e.g. the p10, p35, IE-1 or ΔIE-1 promoters and furtherpromoters described in the above references.

Preferably the nucleic acid construct for expression of the parvoviralRep proteins in insect cells is an insect cell-compatible vector. An“insect cell-compatible vector” or “vector” is understood to a nucleicacid molecule capable of productive transformation or transfection of aninsect or insect cell. Exemplary biological vectors include plasmids,linear nucleic acid molecules, and recombinant viruses. Any vector canbe employed as long as it is insect cell-compatible. The vector mayintegrate into the insect cells genome but the presence of the vector inthe insect cell need not be permanent and transient episomal vectors arealso included. The vectors can be introduced by any means known, forexample by chemical treatment of the cells, electroporation, orinfection. In a preferred embodiment, the vector is a baculovirus, aviral vector, or a plasmid. In a more preferred embodiment, the vectoris a baculovirus, i.e. the construct is a baculoviral vector.Baculoviral vectors and methods for their use are described in the abovecited references on molecular engineering of insect cells.

In another aspect the invention relates to an insect cell that comprisesno more than one type of nucleotide sequence comprising a single openreading frame encoding a parvoviral Rep protein. Preferably the singleopen reading frame encodes one or more of the parvoviral Rep proteins,more preferably the open reading frame encodes all of the parvoviral Repproteins, most preferably the open reading frame encodes the full-lengthRep 78 protein from which preferably at least both Rep 52 and Rep 78proteins may be expressed in the insect cell. It is understood hereinthat the insect cell may comprise more than one copy of the single typeof nucleotide sequence, e.g. in a multicopy episomal vector, but thatthese are multiple copies of essentially one and the same nucleic acidmolecule, or at least nucleic acid molecules that encode one and thesame Rep amino acid sequence, e.g. nucleic acid molecules that onlydiffer between each other due to the degeneracy of the genetic code. Thepresence of only a single type of nucleic acid molecule encoding theparvoviral Rep proteins avoids recombination between homologoussequences as may be present in different types of vectors comprising Repsequences, which may give rise to defective Rep expression constructsthat affect (stability of) parvoviral production levels in insect cells.Preferably, in the insect cell, the nucleotide sequence comprising thesingle open reading frame encoding one or more parvoviral Rep proteinsis part of a nucleic acid construct wherein the nucleotide sequence isoperably linked to expression control sequences for expression in aninsect cell. A further preferred insect cell comprises as a “first”nucleotide sequence a nucleotide sequence as defined above encodingparvoviral Rep proteins, preferably a coding sequence with a suboptimalinitiation codon as defined above, or a nucleic acid construct asdefined above or the insect cell comprises as a “first” nucleic acidconstruct a nucleic acid construct as defined above comprising suchnucleotide sequences.

Any insect cell which allows for replication of a recombinant parvoviral(rAAV) vector and which can be maintained in culture can be used inaccordance with the present invention. For example, the cell line usedcan be from Spodoptera frugiperda, drosophila cell lines, or mosquitocell lines, e.g., Aedes albopictus derived cell lines. Preferred insectcells or cell lines are cells from the insect species which aresusceptible to baculovirus infection, including e.g. Se301, SeIZD2109,SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5Bl-4, MG-1, Tn368, HzAml, Ha2302,Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+® (U.S. Pat. No.6,103,526; Protein Sciences Corp., CT, USA).

A preferred insect cell according to the invention, in addition to theabove described “first” nucleotide sequence or a nucleic acid construct,further comprises:

-   -   a) a second nucleotide sequence comprising at least one        parvoviral inverted terminal repeat (ITR) nucleotide sequence;        and,    -   b) a third nucleotide sequence comprising parvoviral Cap protein        coding sequences operably linked to expression control sequences        for expression in an insect cell.

In the context of the invention “at least one parvoviral ITR nucleotidesequence” is understood to mean a palindromic sequence, comprisingmostly complementary, symmetrically arranged sequences also referred toas “A,” “B,” and “C” regions. The ITR functions as an origin ofreplication, a site having a “cis” role in replication, i.e., being arecognition site for transacting replication proteins such as e.g. Rep78 (or Rep68) which recognize the palindrome and specific sequencesinternal to the palindrome. One exception to the symmetry of the ITRsequence is the “D” region of the ITR. It is unique (not having acomplement within one ITR). Nicking of single-stranded DNA occurs at thejunction between the A and D regions. It is the region where new DNAsynthesis initiates. The D region normally sits to one side of thepalindrome and provides directionality to the nucleic acid replicationstep. An parvovirus replicating in a mammalian cell typically has twoITR sequences. It is, however, possible to engineer an ITR so thatbinding sites are on both strands of the A regions and D regions arelocated symmetrically, one on each side of the palindrome. On adouble-stranded circular DNA template (e.g., a plasmid), the Rep78- orRep68-assisted nucleic acid replication then proceeds in both directionsand a single ITR suffices for parvoviral replication of a circularvector. Thus, one ITR nucleotide sequence can be used in the context ofthe present invention. Preferably, however, two or another even numberof regular ITRs are used. Most preferably, two ITR sequences are used. Apreferred parvoviral ITR is an AAV ITR. For safety reasons it may bedesirable to construct a recombinant parvoviral (rAAV) vector that isunable to further propagate after initial introduction into a cell. Sucha safety mechanism for limiting undesirable vector propagation in arecipient may be provided by using rAAV with a chimeric ITR as describedin US2003148506.

The number of nucleic acid constructs employed in the insect cell forthe production of the recombinant parvoviral (rAAV) vector is notlimiting in the invention. For example, one, two, three, four, five, ormore separate constructs can be employed to produce rAAV in insect cellsin accordance with the methods of the present invention. If fiveconstructs are employed, one construct encodes AAV VP 1, anotherconstruct encodes AAV VP2, yet another construct encodes AAV VP3, stillyet another construct encodes the Rep protein as defined above and afinal construct comprises at least one AAV ITR. If fewer than fiveconstructs are used, the constructs can comprise various combinations ofthe at least one AAV ITR and the VPl, VP2, VP3, and the Rep proteincoding sequences. Preferably, two constructs or three constructs areused, with two constructs being more preferred as described above. Iftwo constructs are used, preferably the insect cell comprises: (a) afirst nucleic acid construct for expression of the Rep proteins asdefined above, which construct further comprises the third nucleotidesequences as defined in (b) above (comprising parvoviral Cap proteincoding sequences operably linked to at least one expression controlsequence for expression in an insect cell; see also below); and (c) asecond nucleic acid construct comprising the second nucleotide sequenceas defined in (a) above (comprising at least one parvoviral/AAV ITRnucleotide sequence). If three constructs are used, preferably the sameconfiguration as used for two constructs is used except that separateconstructs are used for expression of the capsid proteins and forexpression of the Rep proteins. The sequences on each construct can bein any order relative to each other. For example, if one constructcomprises ITRs and an ORF comprising nucleotide sequences encoding VPcapsid proteins, the VP ORF can be located on the construct such that,upon replication of the DNA between ITR sequences, the VP ORF isreplicated or not replicated. For another example, the Rep codingsequences and/or the ORF comprising nucleotide sequences encoding VPcapsid proteins can be in any order on a construct. In is understoodthat also the second, third and further nucleic acid construct(s)preferably are an insect cell-compatible vectors, preferably abaculoviral vectors as described above. Alternatively, in the insectcell of the invention, one or more of the first nucleotide sequence,second nucleotide sequence, third nucleotide sequence, and fourthnucleotide sequence and optional further nucleotide sequences may bestably integrated in the genome of the insect cell. One of ordinaryskill in the art knows how to stably introduce a nucleotide sequenceinto the insect genome and how to identify a cell having such anucleotide sequence in the genome. The incorporation into the genome maybe aided by, for example, the use of a vector comprising nucleotidesequences highly homologous to regions of the insect genome. The use ofspecific sequences, such as transposons, is another way to introduce anucleotide sequence into a genome.

In the invention, the third nucleotide sequence comprising parvoviralcapsid (Cap) protein coding sequences is herein understood to comprisessequences encoding each of the three parvoviral capsid proteins, VPl, -2and -3. The third nucleotide sequence comprising the capsid proteincoding sequences may be present in various forms. E.g. separate codingsequences for each of the capsid proteins VPl, -2 and -3 may used,whereby each coding sequence is operably linked to expression controlsequences for expression in an insect cell. More preferably, however,the third nucleotide sequence comprises a single open reading frameencoding all three of the animal parvoviral (AAV) VPl, VP2, and VP3capsid proteins, wherein the initiation codon for translation of the VPIcapsid protein is a suboptimal initiation codon that is not ATG as e.g.described by Urabe et al. (2002, supra). A suboptimal initiation codonfor the VPl capsid protein may be as defined above for the Rep78protein. More preferred suboptimal initiation codons for the VPI capsidprotein may be selected from ACG, TTG, CTG and GTG, of which CTG and GTGare most preferred. A preferred third nucleotide sequence for theexpression of the capsid proteins further comprises an expressioncontrol sequence comprising a nine nucleotide sequence of SEQ. ID NO:7or a nucleotide sequence substantially homologous to SEQ. ID NO:7,upstream of the initiation codon of the nucleotide sequence encoding theVPI capsid protein. A sequence with substantial identity to thenucleotide sequence of SEQ. ID NO:7 and that will help increaseexpression of VPI is e.g. a sequence which has at least 60%, 70%, 80% or90% identity to the nine nucleotide sequence of SEQ ID NO:7. A furtherpreferred third nucleotide sequence for expression of the capsidproteins further preferably comprises at least one modification of thenucleotide sequence encoding the VP 1 capsid protein selected from amonga C at nucleotide position 12, an A at nucleotide position 21, and a Cat nucleotide position 24 (with reference to position 1 being the firstnucleotide of the translation initiation codon; see SEQ ID NO.I).Elimination of possible false initiation codons for translation of VPlof other serotypes will be well understood by an artisan of skill in theart, as will be the elimination of putative splice sites that may berecognised in insect cells. Various further modifications of VP codingregions are known to the skilled artisan which could either increaseyield of VP and virion or have other desired effects, such as alteredtropism or reduce antigenicity of the virion. These modifications arewithin the scope of the present invention. Preferably the nucleotidesequence of the invention encoding the parvoviral capsid proteins isoperably linked to expression control sequences for expression in aninsect cell, which will at least include a promoter that is active ininsect cells. Such control sequences and further techniques andmaterials (e.g. vectors) for expressing parvoviral capsid proteins ininsect host cells are already described above for the Rep proteins.

In a preferred embodiment of the invention, the second nucleotidesequence present in the insect cells of the invention, i.e. the sequencecomprising at least one parvoviral (AAV) ITR, further comprises at leastone nucleotide sequence encoding a gene product of interest, wherebypreferably the at least one nucleotide sequence encoding a gene productof interest becomes incorporated into the genome of a recombinantparvoviral (rAAV) vector produced in the insect cell. Preferably, atleast one nucleotide sequence encoding a gene product of interest is asequence for expression in a mammalian cell. Preferably, the secondnucleotide sequence comprises two parvoviral (AAV) ITR nucleotidesequences and wherein the at least one nucleotide sequence encoding agene product of interest is located between the two parvoviral (AAV) ITRnucleotide sequences. Preferably, the nucleotide sequence encoding agene product of interest (for expression in the mammalian cell) will beincorporated into the recombinant parvoviral (rAAV) vector produced inthe insect cell if it is located between two regular ITRs, or is locatedon either side of an ITR engineered with two D regions.

The second nucleotide sequence defined herein above may thus comprise anucleotide sequence encoding at least one “gene product of interest” forexpression in a mammalian cell, located such that it will beincorporated into an recombinant parvoviral (rAAV) vector replicated inthe insect cell. Any nucleotide sequence can be incorporated for laterexpression in a mammalian cell transfected with the recombinantparvoviral (rAAV) vector produced in accordance with the presentinvention. The nucleotide sequence may e.g. encode a protein it mayexpress an RNAi agent, i.e. an RNA molecule that is capable of RNAinterference such as e.g. a shRNA (short hairpinRNA) or an siRNA (shortinterfering RNA). “siRNA” means a small interfering RNA that is ashort-length double-stranded RNA that are not toxic in mammalian ells(Elbashir et al., 2001, Nature 411:494-98; Caplen et al., 2001, Proc.Natl. Acad. Sci. USA 98:9742-47). In a preferred embodiment, the secondnucleotide sequence may comprise two nucleotide sequences and eachencodes one gene product of interest for expression in a mammalian cell.Each of the two nucleotide sequences encoding a product of interest islocated such that it will be incorporated into a recombinant parvoviral(rAAV) vector replicated in the insect cell.

The product of interest for expression in a mammalian cell may be atherapeutic gene product. A therapeutic gene product can be apolypeptide, or an RNA molecule (siRNA), or other gene product that,when expressed in a target cell, provides a desired therapeutic effectsuch as e.g. ablation of an undesired activity, e.g. the ablation of aninfected cell, or the complementation of a genetic defect, e.g. causinga deficiency in an enzymatic activity. Examples of therapeuticpolypeptide gene products include CFTR, Factor IX, Lipoprotein lipase(LPL, preferably LPL S447X; see WO 01/00220), Apolipoprotein Al, UridineDiphosphate Glucuronosyltransferase (UGT), Retinitis Pigmentosa GTPaseRegulator Interacting Protein (RP-GRIP), and cytokines or interleukinslike e.g. IL-10.

Alternatively, or in addition as a second gene product, secondnucleotide sequence defined herein above may comprise a nucleotidesequence encoding a polypeptide that serve as marker proteins to assesscell transformation and expression. Suitable marker proteins for thispurpose are e.g. the fluorescent protein GFP, and the selectable markergenes HSV thymidine kinase (for selection on HAT medium), bacterialhygromycin B phosphotransferase (for selection on hygromycin B), Tn5aminoglycoside phosphotransferase (for selection on G418), anddihydrofolate reductase (DHFR) (for selection on methotrexate), CD20,the low affinity nerve growth factor gene. Sources for obtaining thesemarker genes and methods for their use are provided in Sambrook andRussel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NewYork. Furthermore, second nucleotide sequence defined herein above maycomprise a nucleotide sequence encoding a polypeptide that may serve asa fail-safe mechanism that allows to cure a subject from cellstransduced with the recombinant parvoviral (rAAV) vector of theinvention, if deemed necessary. Such a nucleotide sequence, oftenreferred to as a suicide gene, encodes a protein that is capable ofconverting a prodrug into a toxic substance that is capable of killingthe transgenic cells in which the protein is expressed. Suitableexamples of such suicide genes include e.g. the E. coli cytosinedeaminase gene or one of the thymidine kinase genes from Herpes SimplexVirus, Cytomegalovirus and Varicella-Zoster virus, in which caseganciclovir may be used as prodrug to kill the transgenic cells in thesubject (see e.g. Clair et al., 1987, Antimicrob. Agents Chemother.31:844-849).

In another embodiment one of the gene products of interest can be an AAVprotein. In particular, a Rep protein, such as Rep78 or Rep68, or afunctional fragment thereof. A nucleotide sequence encoding a Rep78and/or a Rep68, if present on the genome of a recombinant parvoviral(rAAV) vector of the invention and expressed in a mammalian celltransduced with the vector, allows for integration of the recombinantparvoviral (rAAV) vector into the genome of the transduced mammaliancell. Expression of Rep78 and/or Rep68 in an rAAV-transduced or infectedmammalian cell can provide an advantage for certain uses of therecombinant parvoviral (rAAV) vector, by allowing long term or permanentexpression of any other gene product of interest introduced in the cellby the vector.

In the recombinant parvoviral (rAAV) vectors of the invention the atleast one nucleotide sequence(s) encoding a gene product of interest forexpression in a mammalian cell, preferably is/are operably linked to atleast one mammalian cell-compatible expression control sequence, e.g., apromoter. Many such promoters are known in the art (see Sambrook andRussel, 2001, supra). Contitutive promoters that are broadly expressedin many cell-types, such as the CMV promoter may be used. However, morepreferred will be promoters that are inducible, tissue-specific,cell-type-specific, or cell cycle-specific. For example, forliver-specific expression a promoter may be selected from anal-anti-trypsin promoter, a thyroid hormone-binding globulin promoter,an albumin promoter, LPS (thyroxine-binding globlin) promoter,HCR-ApoCII hybrid promoter, HCR-hAAT hybrid promoter and anapolipoprotein E promoter. Other examples include the E2F promoter fortumor-selective, and, in particular, neurological cell tumor-selectiveexpression (Parr et al., 1997, Nat. Med. 3: 1145-9) or the IL-2 promoterfor use in mononuclear blood cells (Hagenbaugh et al., 1997, J Exp Med;185:2101-10).

AAV is able to infect a number of mammalian cells. See, e.g., Tratschinet al. (1985, 30

Mol. Cell Biol. 5:3251-3260) and Grimm et al. (1999, Hum. Gene Tuer.10:2445-2450). However, AAV transduction of human synovial fibroblastsis significantly more efficient than in similar murine cells, Jenningset al., Arthritis Res, 3:1 (2001), and the cellular tropicity of AAVdiffers among serotypes. See, e.g., Davidson et al. (2000, Proc. Natl.Acad. Sci. USA, 97:3428-3432), who discuss differences among AAV2, AAV4,and AAV5 with respect to mammalian CNS cell tropism and transductionefficiency.

AAV sequences that may be used in the present invention for theproduction of recombinant AAV vectors in insect cells can be derivedfrom the genome of any AAV serotype. Generally, the AAV serotypes havegenomic sequences of significant homology at the amino acid and thenucleic acid levels, provide an identical set of genetic functions,produce virions which are essentially physically and functionallyequivalent, and replicate and assemble by practically identicalmechanisms. For the genomic sequence of the various AAV serotypes and anoverview of the genomic similarities see e.g. GenBank Accession numberU89790; GenBank Accession number J0I90I; GenBank Accession numberAF043303; GenBank Accession number AF0857I6; Chlorini et al. (I997, J.Vir. 7I:6823-33); Srivastava et al. (I983, J. Vir. 45:555-64); Chloriniet al. (I999, J. Vir. 73:1309-13I 9); Rutledge et al. (I998, J. Vir.72:309-3I9); and Wu et al. (2000, J. Vir. 74:8635-47). AAV serotypes I,2, 3, 4 and 5 are preferred source of AAV nucleotide sequences for usein the context of the present invention. Preferably the AAV ITRsequences for use in the context of the present invention are derivedfrom AAVI, AAV2, and/or AAV4. Likewise, the Rep (Rep78 and Rep52) codingsequences are preferably derived from AAVI, AAV2, and/or AAV4. Thesequences coding for the VP I, VP2, and VP3 capsid proteins for use inthe context of the present invention may however be taken from any ofthe known 42 serotypes, more preferably from AAVI, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAVS or AAV9 or newly developed AAV-like particlesobtained by e.g. capsid shuffling techniques and AAV capsid libraries.

AAV Rep and ITR sequences are particularly conserved among mostserotypes. The Rep78 proteins of various AAV serotypes are e.g. morethan 89% identical and the total nucleotide sequence identity at thegenome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82%(Bantel-Schaal et al., I999, J. Virol., 73(2):939-947). Moreover, theRep sequences and ITRs of many AAV serotypes are known to efficientlycross-complement (i.e., functionally substitute) corresponding sequencesfrom other serotypes in production of AAV particles in mammalian cells.US2003I48506 reports that AAV Rep and ITR sequences also efficientlycross-complement other AAV Rep and ITR sequences in insect cells.

The AAV VP proteins are known to determine the cellular tropicity of theAAV virion. The VP protein-encoding sequences are significantly lessconserved than Rep proteins and genes among different AAV serotypes. Theability of Rep and ITR sequences to cross-complement correspondingsequences of other serotypes allows for the production of pseudotypedrAAV particles comprising the capsid proteins of a serotype (e.g., AAV3)and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2).Such pseudotyped rAAV particles are a part of the present invention.

Modified “AAV” sequences also can be used in the context of the presentinvention, e.g. for the production of rAAV vectors in insect cells. Suchmodified sequences e.g. include sequences having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more nucleotide and/or amino acid sequenceidentity (e.g., a sequence having about 75-99% nucleotide sequenceidentity) to an AAVI, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVS or AAV9ITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VPsequences.

Although similar to other AAV serotypes in many respects, AAV5 differsfrom other human and simian AAV serotypes more than other known humanand simian serotypes. In view thereof, the production of rAAV5 candiffer from production of other serotypes in insect cells. Where methodsof the invention are employed to produce rAAV5, it is preferred that oneor more constructs comprising, collectively in the case of more than oneconstruct, a nucleotide sequence comprising an AAV5 ITR, a nucleotidesequence comprises an AAV5 Rep coding sequence (i.e. a nucleotidesequence comprises an AAV5 Rep78). Such ITR and Rep sequences can bemodified as desired to obtain efficient production of rAAV5 orpseudotyped rAAV5 vectors in insect cells. E.g., the start codon of theRep sequences can be modified, VP splice sites can be modified oreliminated, and/or the VPI start codon and nearby nucleotides can bemodified to improve the production of rAAV5 vectors in the insect cell.

In another aspect the invention thus relates to a method for producing arecombinant parvoviral (rAAV) virion (comprising a recombinantparvoviral (rAAV) vector as defined above) in an insect cell.Preferably, the method comprises the steps of: (a) culturing an insectcell as defined in herein above under conditions such that recombinantparvoviral (rAAV) vector is produced; and, (b) recovery of therecombinant parvoviral (rAAV) vector. It is understood here that therecombinant parvoviral (rAAV) vector produced in the method preferablyis an infectious parvoviral or AAV virion that comprise the recombinantparvoviral (rAAV) vector nucleic acids. Growing conditions for insectcells in culture, and production of heterologous products in insectcells in culture are well-known in the art and described e.g. in theabove cited references on molecular engineering of insects cells.

Preferably the method further comprises the step ofaffinity-purification of the (virions comprising the) recombinantparvoviral (rAAV) vector using an anti-AAV antibody, preferably animmobilised antibody. The anti-AAV antibody preferably is an monoclonalantibody. A particularly suitable antibody is a single chain camelidantibody or a fragment thereof as e.g. obtainable from camels or llamas(see e.g. Muyldermans, 2001, Biotechnol. 74:277-302). The antibody foraffinity-purification of rAAV preferably is an antibody thatspecifically binds an epitope on a AAV capsid protein, wherebypreferably the epitope is an epitope that is present on capsid proteinof more than one AAV serotype. E.g. the antibody may be raised orselected on the basis of specific binding to AAV2 capsid but at the sametime also it may also specifically bind to AAVI, AAV3 and AAV5 capsids.

In a further aspect the invention relates to a rAAV virion produced inthe above described methods of the invention, using the nucleic acidconstructs and cells as defined above. Preferably the rAAV virioncomprises in its genome at least one nucleotide sequence encoding a geneproduct of interest, whereby the at least one nucleotide sequence is nota native AAV nucleotide sequence, and whereby in the stoichiometry ofthe AAV VPl, VP2, and VP3 capsid proteins the amount of VPI: (a) is atleast 100, 105, 110, 120, 150, 200 or 400% of the amount of VP2; or (b)is at least 8, 10, 10.5, 11, 12, 15, 20 or 40% of the amount of VP3; or(c) is at least as defined in both (a) and (b). Preferably, the amountof VPl, VP2 and VP3 is determined using an antibody recognising anepitope that is common to each of VPI, VP2 and VP3. Various immunoassaysare available in the art that will allow quantify the relative amountsof VPI, VP2 and/or VP3 (see e.g. Using Antibodies, E. Harlow and D.Lane, 1999, Cold Spring Harbor Laboratory Press, New York). An suitableantibody recognising an epitope that is common to each of the threecapsid proteins is e.g. the mouse anti-Cap Bl antibody (as iscommercially available from Progen, Germany). A preferred rAAV virionaccording to the invention is a virion comprising in its genome at leastone nucleotide sequence encoding a gene product of interest, whereby theat least one nucleotide sequence is not a native AAV nucleotidesequence, and whereby the AAV virion comprises a VPl capsid proteincomprises a leucine or a valine at amino acid position 1. A morepreferred AAV virion according to the invention has the ratio's ofcapsid proteins as defined above and comprises a VP1 capsid proteincomprises a leucine or a valine at amino acid position 1.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A) Organisation of Rep expression in the wild type AAV genome.The Rep78 and Rep 52 genes are expressed from respectively the P5 andP19 promoter. Expression of Rep68 and Rep40 (which are the splicedvariants of resp. Rep78 and Rep52) are not shown. Both expression unitscontain a ATG-initiation site.

B) The construct of the invention has the Rep ORF under the control of asingle promoter (e.g. the polyhedron (PolH) promoter). This promoterdrives the expression of both Rep78 and Rep52 because the Rep78initiation codon ATG is converted to the alternate ACG initiation codonand partially skipped by the ribosome.

C) The original construct by Urabe et al. (2002, supra) drives Rep78 andRep52 independently from two different promoters (resp. MEI and polH).

FIG. 2: Western blot analysis of Rep proteins expressed from recombinantbaculovirus that was passaged 5 times on insect cells. The originalbaculovirus designed by Urabe et al., 2002 (original REP/Bae-to-Bae)results in a slow decrease of Rep78/52 expression over 5 passages. Theexpression unit for Rep78 and 52 designed by Urabe et al., 2002 insertedin baculovirus backbone PSC (original REP/PSC) also results in adecrease of Rep78/52 expression following passaging on insect cells.However, the baculovirus with the REP expression unit containing the ACGinitiation codon in the PSC backbone (REP-ACG/PSC) results in stableexpression of Rep78/52 over at least 5 passages. Western blot analysiswas performed as described in Example 1.1.3.

FIG. 3: Results of Table 1 plotted in a graph.

FIG. 4: Comparison of the stabilities of various rAAV constructs ininsect cells. rAAV production in SF+ cells was performed as describedabove in Example 1. For all productions the ITR containing baculovirusand the capsid gene containing baculovirus were identical, the passagenumber was the same as the Rep gene containing baculoviruses. 4different Rep gene containing baculoviruses were used: 1) TheREP-ACG/PSC, 2) SLR: the original construct by Urabe et al. (2002,supra), 3) Rep52+Rep78(B2B): Two separate Bae-to-Bae baculoviruses, onecontaining the Rep 78 gene and the other one containing the Rep 52 gene.4) Rep52+Rep78(PSC): Two separate protein sciences baculoviruses onecontaining the Rep 78 gene and the other one containing the Rep 52 gene.

FIG. 5: Stability of the REP-ACG/PSC baculovirus constructs up topassage 8. rAAV productions in SF+ cells were performed as described inExample 1.

FIG. 6: Comparison of the effect of passage effect on rep proteinexpression of the original construct from Urabe et al. (2002, supra)with a REP-ACG I PSC construct in accordance with the invention. Thebaculovirus passages and the western blot were done as described inExample 1. During a normal passage of the rep baculoviruses, sampleswere taken at 40 hours after addition of the baculoviruses to the SFcells and western blot was performed.

EXAMPLES Example 1: Rep

1.1. Constructs

1.1.1 Baculovirus Plasmid Construction

In order to express Rep78 and Rep52 from a sole bicistronic messengerRNA, the ATG initiation codon of Rep78 situated on the expression vectorpFastBacDualSLR (Urabe et al., 2002, supra) was converted to ACG. Theupstream primer used was:

BamHI (SEQ ID NO. 8) 5′-cgcggatcctgttaagACGGCGGGGTTTTACGAGATTGTGATTAAGGTC-3′ PRIMER SEQUENCE forward

The 3′-primer that was used in the PCR reaction was flanking the REP78gene and contains a XbaI site (TCTAGA):

XbaI (SEQ ID NO. 9) 5′-AGGCTCTAGATTCGAAAGCGGCCCG-3′PRIMER SEQUENCE reverse

The sequence between the above-mentioned primer set was amplified by PCR(reaction volume 50 μl; Ix Pfx Amp. Buffer, 0.3 mM dNTP's, 1 mM MgS04,150 mM primer forw., 150 mM primer rev., 2× enhancer solution, template50 ng (pFastBacDualSLR), 1 U Platinum Pfx (Invitrogen, Carlsbad, Calif.,USA) using the following protocol: 1 cycle of 95° C., 5 min; 35

cycles of 95° C., 15 sec; 55° C., 30 sec; 72° C., 2 min; 1 cycle of 72°C., 10 min; 4° C., for ever). The PCR product was cloned in PCR-bluntII-TOPO using the Zero Blunt TOPO PCR cloning kit (Invitrogen). TheRep78 was subcloned into pFastBacDual (Invitrogen) using the restrictionsites SpeI and XbaI. The mutated Rep expression cassette was finallycloned (using restriction enzymes BstZl 7I and AvrII) into thebaculovirus expression construct (cut open with EcoRV and XbaI) pPSClO(Protein Sciences Corporation, Meriden, Conn., USA). The sequenceanalysis of the construct was verified by Baseclear, Leiden, theNetherlands.

1.1.2 Recombinant Baculovirus Production

Recombinant baculoviruses derived from the Autographa californicanuclear polyhydrosis vims (AcNPV) were produced using the GeneXpressBaculoKIT (Protein Sciences Corporation). Transfection was performed asfollows: in a round bottom 14 ml tube 200 μl GRACE medium was mixed with6 μl cellfectine (Invitrogen), and in a eppendorf tube 200 μl GRACEmedium was mixed with 50 μl viral DNA (protein sciences) and 2 μgtransfer plasmid (REP). The contents from the eppendorf tube were addedto the tube and mixed carefully. After an incubation period of 30minutes at RT 1,300 μl GRACE was added to the transfection mix.

Insect cells in a T25 flask were washed with GRACE medium and thetransfection mixture was added dropwise to the cell layer. After anincubation of 6 hours at 28° C. SF900II serum supplemented with I0% FBSwas added carefully and the T25 flask was put in a 28° C. stove for 5days after which the recombinant baculovirus was harvested.

1.1.3 Western Blot Analysis

Insect cells (SF+) were infected with baculovirus-REP. At I6, 40, and 64hours post-infection cells a sample was taken and cells were lysed byadding O. IV I0×TRIS lysis buffer (1.5M NaCl, 0.5M TRIS, O.O IM MgCl, I% TRITON X-IOO, pH8.5, filter sterilised) and incubated at 28° C. for 30minutes in a shaker (Innova 44, New Brunswick). Free DNA and RNA wasdegraded by incubation with benzonase at 37° C. for 30 minutes. Celllysate was centrifuged (1,900×g; 15 min; 4° C.). NuPAGE LDS samplebuffer (4×, Invitrogen) was added to a sample of the supernatant and wasloaded onto a 4-12% Bis-Tris gel (120V). Proteins were blotted onto aPVDF membrane (BioRad) for 30 minutes, IOV (Semidry blotting). Westernimmunochemistry was performed by blocking the membrane withSuperblock-PBS blocking buffer (PIERCE) and subsequent incubation withmouse anti-Rep (303.9, Progen, Germany; dilution 1:50) and rabbitanti-mouse-HRP (DAKO, dilution I:500). The Rep-proteins were visualizedby chemoluminescent staining with lumi-light plus Western-blottingsubstrate (Roche).

1.2 Results

The performance of the newly designed Rep-construct of the invention(REP-ACG I PSC) was compared with the original Rep constructs in both I)PSC baculovirus backbone and in 2) Bae-to-Bae baculovirus backbone(Urabe et al., 2002). All three constructs were serially passaged untilpassage 5. AAVI-LPL production experiments were performed using thepassage 2, 3, 4 and 5 Rep-constructs in combination with an AAV-LPL anda AAV-Cap recombinant baculovirus of respectively passage 2, 3, 4 and 5(AAV-LPL and AAV-Cap recombinant Baculovirus used here are describedbelow in Example 2). AAVI-LPL production yields were determined by qPCRand are shown in Table I. The original baculovirus designed by Urabe etal., 2002 (original REP/Bae-to-Bae) results in a fast decrease of AAVproduction over 5 passages. The expression unit for Rep designed byUrabe et al., 2002 inserted in baculovirus backbone PSC (original REP IPSC) also results in a decrease of AAV production following passaging oninsect cells. However, the baculovirus with the REP expression unitcontaining the ACG initiation codon in the PSC backbone (REP-ACG I PSC)results in stable AAV production over at least 5 passages. Therefore,reproducible production yields of AAV-LPL over several passages (e.g. 2to 5) were only obtained using baculoviruses containing the REP-ACGconstruct.

TABLE 1 Production of rAAV virions using the baculovirus constructs ofseveral passages: original REP/ REP-ACG/PSC original REP/Bae-to- passagePSC μg/ml μg/ml Bae μg/ml 2 5.38E+09 3.04E+09 3.62E+10 3 9.57E+094.77E+09 7.28E+09 4 1.66E+09 7.81E+09 7.59E+08 5 7.35E+08 9.90E+092.03E+08 Sf9 cells were infected with three recombinant baculovirusesencoding a LPL-vector unit of passage 2, 3, 4 or 5 , a Rep-expressionunit of passage 2, 3, 4 or 5 and a Cap-expression unit of passage 2, 3,4 or 5. After three days cells were harvested and AAV yields (vectorgenomes per ml; vg/ml) were determined by qPCR.

TABLE 2 Q-PCR performed on the various Bae-Rep constructs followingpassaging on insect cells (Passage 2-5). titer (gc's/ml) Ratio Ratio ORFRep78 Rep52 ORF/Rep7 ORF/Rep original REP/Bac-to-Bac 1.4E+09 2.2E+082.4E+08 6.42 5.82 original REP/Bac-to-Bac 6.4E+08 5.6E+07 5.0E+07 11.4312.93 original REP/Bac-to-Bac 2.1E+09 7.1E+07 6.5E+07 29.47 32.02original REP/Bac-to-Bac 1.7E+09 3.2E+07 2.5E+07 53.68 69.67 REP-ACG/PSC(C4) P2 3.0E+09 2.7E+09 2.9E+09 1.11 1.04 REP-ACG/PSC (C4) P3 2.3E+092.0E+09 2.2E+09 1.11 1.05 REP-ACG/PSC (C4) P4 2.5E+09 2.2E+09 2.3E+091.13 1.08 REP-ACG/PSC (C4) P5 2.7E+09 2.1E+09 2.5E+09 1.26 1.07REP-ACG/PSC (A3) P2 2.5E+09 2.2E+09 2.5E+09 1.18 1.00 REP-ACG/PSC (A3)P3 4.2E+09 3.9E+09 4.0E+09 1.08 1.04 REP-ACG/PSC (A3) P4 2.7E+09 2.4E+092.5E+09 1.10 1.05 REP-ACG/PSC (A3) P5 1.5E+09 1.5E+09 1.5E+09 1.03 0.98original REP/Bae-to-Bae 1.0E+09 1.1E+09 1.1E+09 0.95 0.87 originalREP/Bae-to-Bae 7.1E+08 6.7E+08 8.1E+08 1.07 0.88 original REP/Bae-to-Bae1.3E+08 1.1E+08 1.3E+08 1.18 1.03 original REP/Bae-to-Bae 1.3E+085.3E+07 6.9E+07 2.34 1.82

Table 2 shows the results of a quantitative PCR (Q-PCR) assay that wasdesigned for the Rep-expression unit in the recombinant baculovirusesand for a flanking baculovirus ORF (gene copies per ml; gc's/ml). Theratio between the Q-PCR values determines the presence of deletions inthe Rep-baculovirus. A ratio of I theoretically means that allbaculoviruses in the batch contain a recombinant Rep78 or 52-sequence.The original baculovirus designed by Urabe et al., 2002 (originalREP/Bae-to-Bae) shows significant amounts of the recombinant baculovirusat passage 5 have deletions in the Rep sequences. The expression unitfor Rep78 and 52 designed by Urabe et al., 2002 inserted in baculovirusbackbone PSC (original REP I PSC) shows a very early and dramatic lossof recombinant baculovirus. However, the baculovirus with the REPexpression unit containing the ACG initiation codon in the PSC backbone(REP-ACG/PSC) (clone C4 and A3) show stable recombinant baculovirusesover at least 5 passages.

Example 2: Cap Constructs

2.1.1 Baculovirus Plasmid Construction

In order to express VPI, 2, 3 from a sole polycistronic messenger RNA,the baculovirus-AAV-Cap construct was designed as described by (Urabe etal., 2002, supra). Briefly, the ATG initiation codon of VPI was mutatedto ACG. A potential ATG initiation codon at position II has been changedto ACG. The splice acceptor site downstream of the VPI initiation codonwas destroyed (mutation at position 2I and 24). The mutated Capexpression cassette was cloned into a baculovirus expression construct;pFastBacDual (pFBDAAVIVPmI I) with BamHl/StuI restriction sites. Thisplasmid (pFBDAAVIVPmI I) was the starting material for introduction ofalternate initiation codons for VPI. The forward primer used by Urabe etal. (2002, supra) in order to introduce the mentioned mutations was:

(SEQ ID NO. 1) BamHI             1      11     21 245′-cgcggat cctgttaagACGGCTGCCGACGGTTATCTACCCGATTG GCTC-3′

The following forward primers were used to make the expressionconstructs using pFBDAAVIVPmI I (Urabe et al., 2002, supra) as startingmaterial:

(SEQ ID NO. 2) 5′-cgcggatcctgttaagTTGGCTGCCGACGGTTATCTACCCGATTGGC TC-3′(SEQ ID NO. 3) 5′-cgcggatcctgttaagATTGCTGCCGACGGTTATCTACCCGATTGGC TC-3′(SEQ ID NO. 4) 5′-cgcggatcctgttaagGTGGCTGCCGACGGTTATCTACCCGATTGGC TC-3′(SEQ ID NO. 5) 5′-cgcggatcctgttaagCTGGCTGCCGACGGTTATCTACCCGATTGGC TC-3′

The backward-primer that was used in the PCR reactions with the aboveforward primers was directed to position 230 bp downstream of the VPIinitiation codon and contains a unique Stu I site (AGGCCT).

(SEQ ID NO. 6) 5′-GTCGTAGGCCTTGTCGTGCTCGAGGGCCGC-3′

Fragments were amplified with the above-mentioned sets of forward andbackward primer pairs by PCR. Following digestion of PCR products withBamHI and Stul the PCR products were subcloned into the BamHI/Stul sitesof pFBDAA VIvpmI I resulting in the various to be testedbaculovirus-AAV-Cap constructs. DNA constructs were verified by sequenceanalysis at Baseclear, Leiden, the Netherlands.

2.1.2 Recombinant Baculovirus Production

Recombinant baculoviruses derived from the Autographa californicanuclear polyhydrosis virus (AcNPV) were produced using the Bae-to-Baebaculovirus expression system (Invitrogen). rBac-Cap was amplified byinfecting 2×10⁶ Sf9 cells per ml at an moi of 0.1. Three days afterinfection the cells were spun down and the supernatant containing thevirus recovered.

2.1.3 Recombinant AAV Production

rAAV batches were produced using three recombinant baculovirusesaccording to Urabe et al., 2002. However, for this study one baculovirusharboured an expression construct for the LPL^(s447x)-transgene. Thetherapeutically active agent expressed from the transgene is a naturallyoccurring variant of human lipoprotein lipase, a single chainpolypeptide of 448 amino acids.

The LPL^(s447x) variant has a deletion of two amino acids at theC-terminus of the protein. The second baculovirus harboured anexpression construct for the AAV replication genes, Rep 78 and Rep 52.The third baculovirus harboured the AAVI capsid sequence with either anACG or a TTG, CTG, GTG initiation codon for VPI.

Mammalian-rAAV batches produced with the plasmid-transfection systemwere produced according to Grimm et al., 1998 (Novel tools forproduction and purification of recombinant adeno-associated virusvectors. Hum Gene Ther. I998 Dec. 10; 9(I8):2745-60).

2.1.3 Western Blot Analysis

Insect cells were infected with baculovirus-Cap. At three dayspost-infection cells were centrifuged (3,000 g; I5 min). The supernatantwas filtered through a 0.22 um Millex filter. NuPAGE LDS sample buffer(Invitrogen) was added to a sample of the supernatant and was loadedonto a 4-I2% Bis-Tris gel. The gel was run at IOOV. Proteins wereblotted onto a nitrocellulose membrane (BioRad) for I hr, lOOV, 350 mA.Western immunochemistry was performed by blocking the membrane with I %marvel, dried skimmed milk and subsequently incubation with mouseanti-Cap (BI from Progen, Germany; dilution I:50) and rabbitanti-mouse-HRP (DAKO, dilution I:I00). VPI, 2 and 3 were visualized bychemoluminescent staining with lumi-light plus Western-blottingsubstrate (Roche).

2.1.4 Biochemical Measurements

Human LPL^(s) ⁴⁴⁷ ^(x) activity was assayed as previously describedusing a radioactive trioleoylglycerol emulsion substrate (Nilsson-Ehleand Scholtz, 1976). Human LPLs⁴⁴⁷x immunoreactive mass was assayed usinga sandwich ELISA with chicken IgY and mouse 5D2 anti-hLPL antibodies(Liu et al., 2000). Plasma triglyceride levels were measured by usingcommercial kits following manufacturer protocols (Boehringer Mannheim,#450032).

2.2 Results

2.2.1 Construction of Recombinant Baculovirus

In order to introduce different alternate initiation codons for VPlexpression in the baculovirus plasmid designed by Urabe et al. (2002,supra) a series of upstream primers were designed containing a BamHIrestriction site and either a TTG, ATT, GTG or CTG codon in place of theACG initiation codon of VPI.PCR using these primers in combination witha downstream primer containing a Stul site resulted in amplifiedfragments that were subcloned into the BamHVStuI site of pFBDVPml 1(Bae-Cap). The resulting baculovirus plasmids were used for thepreparation of recombinant baculoviruses using the Bae-to-Baebaculovirus expression system. The prepared recombinant baculoviruseswere infected on insect cells in order to produce AAV capsids. At threedays following infection viral protein expression of the differentbaculovirus batches were determined on Western blots. From the Westernblots it became clear that the baculovirus construct containing the TTGinitiation codon for VP1 expressed this protein to a higher levelcompared to the previously used ACG initiation codon. The ratio betweenVPl and VP2 using the TTG codon was found to be 1:1 which is similar towhat is reported for wild type AAV (not shown).

2.2.2 Infection of rAAV Batches on Cells in Culture

In order to investigate the infectivity of the AAV capsids derived fromrecombinant baculoviruses with the TTG initiation codon rAAV wasgenerated. Also a rAAV batch was generated by plasmid transfection onmammalian HEK293 cells. A vector genome titer of both rAAV batches wasdetermined by qPCR. This titer was used to infect HEK 293 cells in amicrotiter plate at an increasing moi. At two days following infectionan quantitative assay (LPL^(s) ⁴⁴⁷ ^(x)-mass assay) for the transgeneproduct (LPL^(s) ⁴⁴⁷ ^(x)) was performed on the medium of the infectedcells. The assay showed that the amount of LPLs⁴⁴⁷x produced bybaculovirus-produced rAAV was similar to the LPL produced by theplasmid-produced rAAV (not shown).

2.2.3 Injection of rAAV Batches in Mice

The rAAV batches produced with the baculovirus-production system andwith the conventional mammalian plasmid-production system were injectedintramuscularly in mice to follow LPLs⁴⁴⁷x protein activity andtriglyceride fasting in vivo. At 3 days, 7 days and at 2 weeks followinginjection blood samples were taken and evaluated. Between 3 and 7 dayspost vims administration blood-plasma sampled from both mice injectedwith mammalian-rAAV and one mouse injected with baculo-rAAV was turnedfrom milky to completely clear. Blood plasma derived from onebaculo-rAAV-injected mouse remained relatively milky however fat levelwas clearly reduced. Triglyceride levels were lowered respectively ofall treated mice (not shown). On day 14 TG levels in both mammalian-AAVand baculovirus-(TTG)-AAV treated mice TG levels were reduced for 96%.Plasma samples taken at two weeks after vims administration showed thatthe LPLs⁴⁴⁷x-activity of the mice treated with baculovirus-AAV andmammalian-AAV was similar (not shown).

Example 3: Stability of rAAV Constructs with Modified Rep 78 InitiationCodon in Insect Cells

3.1 Comparison of the Stabilities of Various rAAV Constructs in InsectCells

rAAV productions in SF+ cells were performed as described above inExample 1. For all productions the ITR containing baculovirus and thecapsid gene containing baculovirus were identical, the passage numberwas the same as the Rep gene containing baculoviruses. 4 different Repgene containing baculovirus were used: 1) The REP-ACG/PSC, 2) SLR: theoriginal construct by Urabe et al. (2002, supra), 3) Rep52+Rep78(B2B):Two separate Bae-to-Bae baculoviruses, one containing the Rep 78 geneand the other one containing the Rep 52 gene. 4) Rep52+Rep78(PSC): Twoseparate protein sciences baculoviruses one containing the Rep 78 geneand the other one containing the Rep 52 gene.

Results are shown in FIG. 4 at fifth baculovirus passage rAAV productionis already improved by more than a factor 10 using a REP-ACG/PSC inaccordance with invention as compared to the original Rep construct andcompared to the split Rep constructs.

3.2 Stability of the Baculovirus Constructs Up to Passage 8

rAAV productions in SF+ cells were performed as described in Example 1.For all productions the ITR containing baculovirus and the capsid genecontaining baculovirus were identical, the passage number was the sameas the REP-ACG/PSC baculovirus. Results are shown in FIG. 5. TheREP-ACG/PSC baculovirus is stable to at least passage 8. rAAV productiontiters of REP-ACG/PSC are stable up to at least 8th passage of thebaculovirus.

3.3 Passage Effect on Rep Protein Expression

The effect of passage number on the expression of Rep protein for theoriginal construct from Urabe et al. (2002, supra) was compared to aREP-ACG I PSC construct in accordance with the invention. Thebaculovirus passages and the western blot were done as described inExample 1. During a normal passage of the rep baculoviruses, sampleswere taken at 40 hours after addition of the baculoviruses to the SFcells and western blot was performed. FIG. 6 clearly shows diminishedRep expression in higher passages compared to earlier passages for theoriginal Urabe construct (SLR), while the Rep expression in theREP-ACG/PSC construct stays the same in the higher passages compared tothe lower ones.

1. A method of producing a recombinant parvoviral viron in an insectcell comprising, transfecting and insect cell with a nucleotide sequenceencoding parvoviral Rep proteins Rep78 and Rep52, the nucleotidesequence comprising a Rep78 coding sequence and a Rep52 coding sequence,the Rep78 coding sequence and the Rep52 coding sequence being operablylinked to expression control sequences for expression of both a Rep78protein and a Rep52 protein in an insect cell; wherein the Rep52 codingsequence is comprised within the Rep78 coding sequence, and wherein amolar ratio of Rep78 and Rep52 in the range of 1:10 to 10:1 is producedwhen the nucleotide sequence is expressed in the insect cell.
 2. Themethod of claim 1, wherein the expression control sequences comprise apromoter or an initiation codon.
 3. The method of claim 2, wherein theinitiation codon is a suboptimal initiation codon.
 4. The method ofclaim 1, wherein the nucleotide sequence is comprised within a nucleicacid construct.
 5. The method of claim 4, wherein the nucleic acidconstruct comprises a second nucleotide sequence comprising at least oneparvoviral inverted terminal repeat (ITR) sequence and a thirdnucleotide sequence comprising at least one parvoviral Cap proteinencoding sequence operably linked to an expression control sequence forexpressing the at least one Cap protein in an insect cell.
 6. The methodof claim 4, wherein the nucleic acid construct comprises onepolyadenylation sequence at its 3′-end.
 7. The method of claim 4,wherein the nucleic acid construct is inserted into a baculovirusvector.
 8. The method of claim 1, wherein the insect cell is selectedfrom the group consisting of Se301, Seizd2109, Seucr1, sf9, Sf900+,Sf21, Bti-Tn-5b1-4, Mg-1, Tn368, Hzam1, Ha2302, Hz2e5, High five andExpress+.
 9. A method of producing a recombinant parvoviral viron in aninsect cell comprising, transfecting and insect cell with a nucleic acidconstruct encoding parvoviral Rep protein Rep78 and Rep52, the nucleicacid construct comprising: (a) a biscistronic nucleic acid encoding aparvovirus Rep78 protein and a parvovirus Rep52 protein, wherein thenucleic acid sequence encoding the Rep78 protein overlaps the nucleicacid sequence encoding the Rep52 protein, such that the nucleic acidsequence encoding the parvoviral Rep52 protein is not repeated in thebiscistronic nucleic acid; (b) a first expression control sequence thatis active in an insect cell operably linked to the sequence encodingRep78 protein; (c) a second expression control sequence that is activein an insect cell operably linked to the sequence encoding Rep52protein; wherein both the parvoviral Rep78 protein and the parvoviralRep52 protein are produced when the nucleic acid construct is expressedin the insect cell.
 10. The method of claim 9, wherein the parvoviralRep78 protein and the parvoviral Rep52 protein are produced in a molarratio in the range of 1:10 to 10:1.
 11. The method of claim 9, whereinthe first expression control sequence comprises an initiation codon. 12.The method of claim 11, wherein the initiation codon is a suboptimalinitiation codon.
 13. The method of claim 9, wherein the first andsecond expression control sequences comprise a promoter.
 14. The methodof claim 9, wherein the second expression control sequence comprises aninternal ribosome entry site (IRES).
 15. The method of claim 9, whereinthe nucleic acid construct comprises one polyadenylation sequence at its3′-end.
 16. The method of claim 9, wherein the nucleic acid construct isinserted into a baculovirus vector.
 17. The method of claim 9, whereinthe insect cell is selected from the group consisting of Se301,Seizd2109, Seucr1, sf9, Sf900+, Sf21, Bti-Tn-5b1-4, Mg-1, Tn368, Hzam1,Ha2302, Hz2e5, High five and Express+.